Compositions and methods for treating bone deficit conditions

ABSTRACT

Compounds containing two aromatic systems covalently linked through a linker containing one or more atoms, or &#34;linker&#34; defined as including a covalent bond per se so as to space the aromatic systems at a distance 1.5-15 Å, are effective in treating conditions associated with bone deficits. The compounds can be administered to vertebrate subjects alone or in combination with additional agents that promote bone growth or that inhibit bone resorption. They can be screened for activity prior to administration by assessing their ability to effect the transcription of a reporter gene coupled to a promoter associated with a bone morphogenetic protein and/or their ability to stimulate calvarial growth in model animal systems.

This application is a CON of U.S. application Ser. No. 08/736,319, filedOct. 23, 1996, now abandoned. The disclosure of this application isincorporated herein by reference.

TECHNICAL FIELD

The invention relates to compositions and methods for use in limitingundesired bone loss in a vertebrate at risk of such bone loss, intreating conditions that are characterized by undesired bone loss or bythe need for bone growth, in treating fractures, and in treatingcartilage disorders. More specifically, the invention concerns the useof specific classes of compounds identified or characterized by a highthroughput screening assay.

BACKGROUND ART

Bone is not a static tissue. It is subject to constant breakdown andresynthesis in a complex process mediated by osteoblasts, which producenew bone, and osteoclasts, which destroy bone. The activities of thesecells are regulated by a large number of cytokines and growth factors,many of which have now been identified and cloned. Mundy has describedthe current knowledge related to these factors (Mundy, G. R. Clin Orthop324:24-28, 1996; Mundy, G. R. J Bone Miner Res 8:S505-10, 1993).

Although there is a great deal of information available on the factorswhich influence the breakdown and resorption of bone, information ongrowth factors which stimulate the formation of new bone is morelimited. Investigators have searched for sources of such activities, andhave found that bone tissue itself is a storehouse for factors whichhave the capacity for stimulating bone cells. Thus, extracts of bovinebone tissue obtained from slaughterhouses contain not only structuralproteins which are responsible for maintaining the structural integrityof bone, but also biologically active bone growth factors which canstimulate bone cells to proliferate. Among these latter factors aretransforming growth factor B, the heparin-binding growth factors (acidicand basic fibroblast growth factor), the insulin-like growth factors(insulin-like growth factor I and insulin-like growth factor II), and arecently described family of proteins called bone morphogenetic proteins(BMPs). All of these growth factors have effects on other types ofcells, as well as on bone cells.

The BMPs are novel factors in the extended transforming growth factor Bsuperfamily. They were first identified by Wozney J. et al. Science(1988) 242: 1528-34, using gene cloning techniques, following earlierdescriptions characterizing the biological activity in extracts ofdemineralized bone (Urist M. Science (1965) 150: 893-99). RecombinantBMP2 and BMP4 can induce new bone formation when they are injectedlocally into the subcutaneous tissues of rats (Wozney J. Molec ReprodDev (1992) 32:160-67). These factors are expressed by normal osteoblastsas they differentiate, and have been shown to stimulate osteoblastdifferentiation and bone nodule formation in vitro as well as boneformation in vivo (Harris S. et al. J. Bone Miner Res (1994) 9:855-63).This latter property suggests potential usefulness as therapeutic agentsin diseases which result in bone loss.

The cells which are responsible for forming bone are osteoblasts. Asosteoblasts differentiate from precursors to mature bone-forming cells,they express and secrete a number of enzymes and structural proteins ofthe bone matrix, including Type-1 collagen, osteocalcin, osteopontin andalkaline phosphatase (Stein G. et al. Curr Opin Cell Biol (1990)2:1018-27; Harris S. et al. (1994), supra). They also synthesize anumber of growth regulatory peptides which are stored in the bonematrix, and are presumably responsible for normal bone formation. Thesegrowth regulatory peptides include the BMPs (Harris S. et al. (1994),supra). In studies of primary cultures of fetal rat calvarialosteoblasts, BMPs 1, 2, 3, 4, and 6 are expressed by cultured cellsprior to the formation of mineralized bone nodules (Harris S. et al.(1994), supra). Like alkaline phosphatase, osteocalcin and osteopontin,the BMPs are expressed by cultured osteoblasts as they proliferate anddifferentiate.

Although the BMPs are potent stimulators of bone formation in vitro andin vivo, there are disadvantages to their use as therapeutic agents toenhance bone healing. Receptors for the bone morphogenetic proteins havebeen identified in many tissues, and the BMPs themselves are expressedin a large variety of tissues in specific temporal and spatial patterns.This suggests that BMPs may have effects on many tissues other thanbone, potentially limiting their usefulness as therapeutic agents whenadministered systemically. Moreover, since they are peptides, they wouldhave to be administered by injection. These disadvantages impose severelimitations to the development of BMPs as therapeutic agents.

There is a plethora of conditions which are characterized by the need toenhance bone formation. Perhaps the most obvious is the case of bonefractures, where it would be desirable to stimulate bone growth and tohasten and complete bone repair. Agents that enhance bone formationwould also be useful in facial reconstruction procedures. Other bonedeficit conditions include bone segmental defects, periodontal disease,metastatic bone disease, osteolytic bone disease and conditions whereconnective tissue repair would be beneficial, such as healing orregeneration of cartilage defects or injury. Also of great significanceis the chronic condition of osteoporosis, including age-relatedosteoporosis and osteoporosis associated with post-menopausal hormonestatus. Other conditions characterized by the need for bone growthinclude primary and secondary hyperparathyroidism, disuse osteoporosis,diabetes-related osteoporosis, and glucocorticoid-related osteoporosis.In addition, or alternatively, the compounds of the present inventionmay modulate metabolism, proliferation and/or differentiation of normalor aberrant cells or tissues.

There are currently no satisfactory pharmaceutical approaches tomanaging any of these conditions. Bone fractures are still treatedexclusively using casts, braces, anchoring devices and other strictlymechanical means. Further bone deterioration associated withpost-menopausal osteoporosis has been decreased or prevented withestrogens or bisphosphonates.

U.S. Pat. No. 5,280,040 discloses a class of compounds which are3,4-diaryl chromans. These compounds can be considered derivatives of2,3,4 triphenyl butanol, where the hydroxy at the 1-position forms anether with the ortho position of the phenyl group substituted at the4-position of the butanol. The parent 3,4-diaryl chromans do not containnitrogen atoms in the aromatic moieties or their linkers. A preferredcompound, centchroman contains a nitrogen substituent only in one of thesubstituents on a phenyl moiety. These compounds are disclosed in the'040 patent as useful in the treatment of osteoporosis.

The present invention discloses compounds useful for limiting ortreating bone deficit conditions, and for other uses that should beapparent to those skilled in the art from the teachings herein.

DISCLOSURE OF THE INVENTION

The invention provides compounds that can be administered as ordinarypharmaceuticals and have the metabolic effect of enhancing bone growth.The compounds of the invention can be identified using an assay fortheir ability to activate control elements associated with thesefactors. Thus, the invention is directed to methods and compositions forstimulating the growth of skeletal (bone) tissue, which methods andcompositions use, as active ingredients, compounds wherein two aromaticsystems are coupled so as to be spaced apart from each other by about1.5 to about 15 Angstroms. The thus-linked systems (including the linkercoupling them) generally include at least one nitrogen atom.

Therefore, the compounds useful in the invention can be described ashaving the formula Ar¹ -linker-Ar², wherein each of Ar¹ and Ar² isindependently an aromatic system and the linker portion of the formulaspaces Ar¹ and Ar² apart by a distance of approximately 1.5-15Angstroms. Ar¹, Ar² and the linker may optionally be substituted withnon interfering substituents. In the useful compounds, there isgenerally at least one nitrogen atom in either Ar¹, Ar² and/or thelinker, independent of any substituents thereon. Preferably, thecompounds of the invention contain at least one additional heteroatomselected from the group consisting of N, S and O, independent of anysubstituent.

Thus, in one aspect, the invention is directed to a method to treat acondition in a vertebrate animal characterized by a deficiency in, orneed for, bone growth replacement and/or an undesirable level of boneresorption, which method comprises administering to a vertebrate subjectin need of such treatment an effective amount of a compound of theformula: ##STR1## wherein R¹ is a non-interfering substituent; m is aninteger of 0-4;

each dotted line represents an optional π-bond;

each Z is independently N, NR, O, S, CR or CR₂, where each R isindependently H or alkyl (1-6C);

L is a constrained linker; and

Ar² is a bivalent phenyl, bivalent napthyl, a bivalent aromatic systemcontaining a 6-membered ring or a bivalent aromatic system containing a5-membered ring, all of which may be substituted or unsubstituted.

In other aspects, the invention relates to pharmaceutical compositionsfor use in the method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 gives a schematic representation of the compounds used as activeingredients in the methods of the invention.

FIG. 2 shows the dose response curve for a positive control compound,designated 59-0008.

FIGS. 3 and 4 show illustrative compounds of the invention and theresults obtained with them in an in vitro test.

MODES OF CARRYING OUT THE INVENTION

A rapid throughput screening test for compounds capable of stimulatingexpression of a reporter gene linked to a BMP promoter (a surrogate forthe production of bone morphogenetic factors that are endogenouslyproduced) is described in U.S. application Ser. No. 08/458,434, filedJun. 2, 1995, the contents of which are incorporated herein byreference. This assay is also described as a portion of a study ofimmortalized murine osteoblasts (derived from a mouse expressing atransgene composed of a BMP2 promoter driving expression of T-antigen)in Ghosh-Choudhery, N. et al. Endocrinology (1996) 137.331-39. In thisstudy, the immortalized cells were stably transfected with a plasmidcontaining a luciferase reporter gene driven by a mouse BMP2 promoter(-2736/114 bp), and responded in a dose-dependent manner to recombinanthuman BMP2.

Briefly, the assay utilizes cells transformed permanently or transientlywith constructs in which the promoter of a bone morphogenetic protein,specifically BMP2 or BMP4, is coupled to a reporter gene, typicallyluciferase. These transformed cells are then evaluated for theproduction of the reporter gene product; compounds that activate the BMPpromoter will drive production of the reporter protein, which can bereadily assayed. Over 40,000 compounds have been subjected to this rapidscreening technique, and only a very small percentage are able to elicita level of production of luciferase 5-fold greater than that produced byvehicle. Compounds that activate the BMP promoter share certainstructural characteristics not present in inactive compounds. The activecompounds ("BMP promoter-active compounds" or "active compounds") areuseful in promoting bone or cartilage growth, and thus in the treatmentof vertebrates in need of bone or cartilage growth.

BMP promoter-active compounds can be examined in a variety of otherassays that test specificity and toxicity. For instance, non-BMPpromoters or response elements can be linked to a reporter gene andinserted into an appropriate host cell. Cytotoxicity can be determinedby visual or microscopic examination of BMP promoter- and/or non-BMPpromoter-reporter gene-containing cells, for instance. Alternatively,nucleic acid and/or protein synthesis by the cells can be monitored. Forin vivo assays, tissues may be removed and examined visually ormicroscopically, and optionally examined in conjunction with dyes orstains that facilitate histologic examination. In assessing in vivoassay results. it may also be useful to examine biodistribution of thetest compound, using conventional medicinal chemistry/animal modeltechniques.

As used herein, "limit" or "limiting" and "treat" or "treatment" areinterchangeable terms. The terms include a postponement of developmentof bone deficit symptoms and/or a reduction in the severity of suchsymptoms that will or are expected to develop. The terms further includeameliorating existing bone or cartilage deficit symptoms, preventingadditional symptoms, ameliorating or preventing the underlying metaboliccauses of symptoms, preventing or reversing bone resorption and/orencouraging bone growth. Thus, the terms denote that a beneficial resulthas been conferred on a vertebrate subject with a cartilage, bone orskeletal deficit, or with the potential to develop such deficit.

By "bone deficit" is meant an imbalance in the ratio of bone formationto bone resorption, such that, if unmodified, the subject will exhibitless bone than desirable, or the subject's bones will be less intact andcoherent than desired. Bone deficit may also result from fracture, fromsurgical intervention or from dental or periodontal disease. By"cartilage defect" is meant damaged cartilage, less cartilage thandesired, or cartilage that is less intact and coherent than desired.

Representative uses of the compounds of the present invention include:repair of bone defects and deficiencies, such as those occuring inclosed, open and non-union fractures; prophylactic use in closed andopen fracture reduction; promotion of bone healing in plastic surgery;stimulation of bone ingrowth into non-cemented prosthetic joints anddental implants; elevation of peak bone mass in pre-menopausal women,treatment of growth deficiencies; treatment of peridontal disease anddefects, and other tooth repair processes; increase in bone formationduring distraction osteogenesis; and treatment of other skeletaldisorders, such as age-related osteoporosis, post-menopausalosteoporosis, glucocorticoid-induced osteoporosis or disuse osteoporosisand arthritis. The compounds of the present invention can also be usefulin repair of congenital, trauma-induced or surgical resection of bone(for instance, for cancer treatment), and in cosmetic surgery. Further,the compounds of the present invention can be used for limiting ortreating cartilage defects or disorders, and may be useful in woundhealing or tissue repair.

Bone or cartilage deficit or defect can be treated in vertebratesubjects by administering compounds of the invention which have beenidentified through suitable screening assays and which exhibit certainstructural characteristics. The compositions of the invention may beadministered systemically or locally. For systemic use, the compoundsherein are formulated for parenteral (e.g., intravenous, subcutaneous,intramuscular, intraperitoneal, intranasal or transdermal) or enteral(e.g., oral or rectal) delivery according to conventional methods.Intravenous administration will be by a series of injections or bycontinuous infusion over an extended period. Administration by injectionor other routes of discretely spaced administration will generally beperformed at intervals ranging from weekly to once to three times daily.Alternatively, the compounds disclosed herein may be administered in acyclical manner (administration of disclosed compound; followed by noadministration; followed by administration of disclosed compound, andthe like). Treatment will continue until the desired outcome isachieved. In general, pharmaceutical formulations will include acompound of the present invention in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater, borate-buffered saline containing trace metals or the like.Formulations may further include one or more excipients, preservatives,solubilizers, buffering agents, albumin to prevent protein loss on vialsurfaces, lubricants, fillers, stabilizers, etc. Methods of formulationare well known in the art and are disclosed, for example, in Remington'sPharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton Pa.,1990, which is incorporated herein by reference. Pharmaceuticalcompositions for use within the present invention can be in the form ofsterile, non-pyrogenic liquid solutions or suspensions, coated capsules,suppositories, lyophilized powders, transdermal patches or other formsknown in the art. Local administration may be by injection at the siteof injury or defect, or by insertion or attachment of a solid carrier atthe site, or by direct, topical application of a viscous liquid. Forlocal administration, the delivery vehicle preferably provides a matrixfor the growing bone or cartilage, and more preferably is a vehicle thatcan be absorbed by the subject without adverse effects.

Delivery of compounds herein to wound sites may be enhanced by the useof controlled-release compositions, such as those described in pendingU.S. patent application Ser. No. 07/871,246 (corresponding to WIPOpublication WO 93/20859, which is incorporated herein by reference inits entirety). Films of this type are particularly useful as coatingsfor prosthetic devices and surgical implants. The films may, forexample, be wrapped around the outer surfaces of surgical screws, rods,pins, plates and the like. Implantable devices of this type areroutinely used in orthopedic surgery. The films can also be used to coatbone filling materials, such as hydroxyapatite blocks, demineralizedbone matrix plugs, collagen matrices and the like. In general, a film ordevice as described herein is applied to the bone at the fracture site.Application is generally by implantation into the bone or attachment tothe surface using standard surgical procedures.

In addition to the copolymers and carriers noted above, thebiodegradable films and matrices may include other active or inertcomponents. Of particular interest are those agents that promote tissuegrowth or infiltration, such as growth factors. Exemplary growth factorsfor this purpose include epidermal growth factor (EGF), fibroblastgrowth factor (FGF), platelet-derived growth factor (PDGF), transforminggrowth factors (TGFs), parathyroid hormone (PTH), leukemia inhibitoryfactor (LIF), and insulin-like growth factors (IGFs). Agents thatpromote bone growth, such as bone morphogenetic proteins (U.S. Pat. No.4,761,471; PCT Publication WO 90/11366), osteogenin (Sampath et al.Proc. Natl. Acad. Sci. USA (1987) 84:7109-13) and NaF (Tencer et al. J.Biomed. Mat. Res. (1989) 23: 571-89) are also preferred. Biodegradablefilms or matrices include calcium sulfate, tricalcium phosphate,hydroxyapatite, polylactic acid, polyanhydrides, bone or dermalcollagen, pure proteins, extracellular matrix components andcombinations thereof. Such biodegradable materials may be used incombination with non-biodegradable materials, to provide desiredmechanical, cosmetic or tissue or matrix interface properties.

Alternative methods for delivery of compounds of the present inventioninclude use of ALZET osmotic minipumps (Alza Corp., Palo Alto, Calif.);sustained release matrix materials such as those disclosed in Wang etal. (PCT Publication WO 90/11366); electrically charged dextran beads,as disclosed in Bao et al. (PCT Publication WO 92/03125); collagen-baseddelivery systems, for example, as disclosed in Ksander et al. Ann. Surg.(1990) 211(3):288-94; methylcellulose gel systems, as disclosed in Becket al. J. Bone Min. Res. (1991) 6(11): 1257-65; and alginate-basedsystems, as disclosed in Edelman et al. Biomaterials (1991) 12:619-26.Other methods well known in the art for sustained local delivery in boneinclude porous coated metal protheses that can be impregnated and solidplastic rods with therapeutic compositions incorporated within them.

The compounds of the present invention may also be used in conjunctionwith agents that inhibit bone resorption. Antiresorptive agents, such asestrogen, bisphosphonates and calcitonin, are preferred for thispurpose. More specifically, the compounds disclosed herein may beadministered for a period of time (for instance, months to years)sufficient to obtain correction of a bone deficit condition. Once thebone deficit condition has been corrected, the vertebrate can beadministered an anti-resorptive compound to maintain the corrected bonecondition. Alternatively, the compounds disclosed herein may beadminstered with an anti-resorptive compound in a cyclical manner(administration of disclosed compound, followed by anti-resorptive,followed by disclosed compound, and the like).

In additional formulations, conventional preparations such as thosedescribed below may be used.

Aqueous suspensions may contain the active ingredient in admixture withpharmacologically acceptable excipients, comprising suspending agents,such as methyl cellulose, and wetting agents, such as lecithin,lysolethicin or long-chain fatty alcohols. The said aqueous suspensionsmay also contain preservatives, coloring agents, flavoring agents andsweetening agents in accordance with industry standards.

Preparations for topical and local application comprise aerosol sprays,lotions, gels and ointments in pharmaceutically appropriate vehicleswhich may comprise lower aliphatic alcohols, polyglycols such asglycerol, polyethylene glycol, esters of fatty acids, oils and fats, andsilicones. The preparations may further comprise antioxidants, such asascorbic acid or tocopherol, and preservatives, such as p-hydroxybenzoicacid esters.

Parenteral preparations comprise particularly sterile or sterilizedproducts. Injectable compositions may be provided containing the activecompound and any of the well known injectable carriers. These maycontain salts for regulating the osmotic pressure.

If desired, the osteogenic agents can be incorporated into liposomes byany of the reported methods of preparing liposomes for use in treatingvarious pathogenic conditions. The present compositions may utilize thecompounds noted above incorporated in liposomes in order to direct thesecompounds to macrophages, monocytes, other cells and tissues and organswhich take up the liposomal composition. The liposome-incorporatedcompounds of the invention can be utilized by parenteral administration,to allow for the efficacious use of lower doses of the compounds.Ligands may also be incorporated to further focus the specificity of theliposomes.

Suitable conventional methods of liposome preparation include, but arenot limited to, those disclosed by Bangham, A. D. et al. J Mol Biol(1965) 23:238-252, Olson, F. et al. Biochim Biophys Acta (1979)557:9-23, Szoka, F. et al. Proc Natl Acad Sci USA (1978) 75:4194-4198,Mayhew, E. et al. (1984) 775:169175, Kim, S. et al. Biochim Biophys Acta(1983) 728:339:348, and Mayer, et al. Biochim Biophys Acta (1986)858:161-168.

The liposomes may be made from the present compounds in combination withany of the conventional synthetic or natural phospholipid liposomematerials including phospholipids from natural sources such as egg,plant or animal sources such as phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin,phosphatidylserine, or phosphatidylinositol. Synthetic phospholipidsthat may also be used, include, but are not limited to:dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidycholine, and thecorresponding synthetic phosphatidylethanolamines andphosphatidylglycerols. Cholesterol or other sterols, cholesterolhemisuccinate, glycolipids, cerebrosides, fatty acids, gangliosides,sphingolipids, 1,2-bis(oleoyloxy)-3-(trimethyl ammonio) propane (DOTAP),N-[1-(2,3-dioleoyl) propyl-N,N,N-trimethylammonium chloride (DOTMA), andother cationic lipids may be incorporated into the liposomes, as isknown to those skilled in the art. The relative amounts of phospholipidand additives used in the liposomes may be varied if desired. Thepreferred ranges are from about 60 to 90 mole percent of thephospholipid; cholesterol, cholesterol hemisuccinate, fatty acids orcationic lipids may be used in amounts ranging from 0 to 50 molepercent. The amounts of the present compounds incorporated into thelipid layer of liposomes can be varied with the concentration of theirlipids ranging from about 0.01 to about 50 mole percent.

Using conventional methods, approximately 20 to 30% of the compoundpresent in solution can be entrapped in liposomes; thus, approximately70 to 80% of the active compound is wasted. In contrast, where thecompound is incorporated into liposomes, virtually all of the compoundis incorporated into the liposome, and essentially none of the activecompound is wasted.

The liposomes with the above formulations may be made still morespecific for their intended targets with the incorporation of monoclonalantibodies or other ligands specific for a target. For example,monoclonal antibodies to the BMP receptor may be incorporated into theliposome by linkage to phosphatidylethanolamine (PE) incorporated intothe liposome by the method of Leserman, L. et al. Nature (1980)288:602-604.

Veterinary uses of the disclosed compounds are also contemplated. Suchuses would include limitation or treatment of bone or cartilage deficitsor defects in domestic animals, livestock and thoroughbred horses. Thecompounds described herein can also modify a target tissue or organenvironment, so as to attract bone-forming cells to an environment inneed of such cells.

The compounds of the present invention may also be used to stimulategrowth of bone-forming cells or their precursors, or to inducedifferentiation of bone-forming cell precursors, either in vitro or exvivo. As used herein, the term "precursor cell" refers to a cell that iscommitted to a differentiation pathway, but that generally does notexpress markers or function as a mature, fully differentiated cell. Asused herein, the term "mesenchymal cells" or "mesenchymal stem cells"refers to pluripotent progenitor cells that are capable of dividing manytimes, and whose progeny will give rise to skeletal tissues, includingcartilage, bone, tendon, ligament, marrow stroma and connective tissue(see A. Caplan J. Orthop. Res. (1991) 9:641-50). As used herein, theterm "osteogenic cells" includes osteoblasts and osteoblast precursorcells. More particularly, the disclosed compounds are useful forstimulating a cell population containing marrow mesenchymal cells,thereby increasing the number of osteogenic cells in that cellpopulation. In a preferred method, hematopoietic cells are removed fromthe cell population, either before or after stimulation with thedisclosed compounds. Through practice of such methods, osteogenic cellsmay be expanded. The expanded osteogenic cells can be infused (orreinfused) into a vertebrate subject in need thereof. For instance, asubject's own mesenchymal stem cells can be exposed to compounds of thepresent invention ex vivo, and the resultant osteogenic cells could beinfused or directed to a desired site within the subject, where furtherproliferation and/or differentiation of the osteogenic cells can occurwithout immunorejection. Alternatively, the cell population exposed tothe disclosed compounds may be immortalized human fetal osteoblastic orosteogenic cells. If such cells are infused or implanted in a vertebratesubject, it may be advantageous to "immunoprotect" these non-self cells,or to immunosuppress (preferably locally) the recipient to enhancetransplantation and bone or cartilage repair.

Within the present invention, an "effective amount" of a composition isthat amount which produces a statistically significant effect. Forexample, an "effective amount" for therapeutic uses is the amount of thecomposition comprising an active compound herein required to provide aclinically significant increase in healing rates in fracture repair;reversal of bone loss in osteoporosis; reversal of cartilage defects ordisorders; prevention or delay of onset of osteoporosis; stimulationand/or augmentation of bone formation in fracture non-unions anddistraction osteogenesis; increase and/or acceleration of bone growthinto prosthetic devices; and repair of dental defects. Such effectiveamounts will be determined using routine optimization techniques and aredependent on the particular condition to be treated, the condition ofthe patient, the route of administration, the formulation, and thejudgment of the practitioner and other factors evident to those skilledin the art. The dosage required for the compounds of the invention (forexample, in osteoporosis where an increase in bone formation is desired)is manifested as a statistically significant difference in bone massbetween treatment and control groups. This difference in bone mass maybe seen, for example, as a 5-20% or more increase in bone mass in thetreatment group. Other measurements of clinically significant increasesin healing may include, for example, tests for breaking strength andtension, breaking strength and torsion, 4-point bending, increasedconnectivity in bone biopsies and other biomechanical tests well knownto those skilled in the art. General guidance for treatment regimens isobtained from experiments carried out in animal models of the disease ofinterest.

The dosage of the compounds of the invention will vary according to theextent and severity of the need for treatment, the activity of theadministered compound, the general health of the subject, and otherconsiderations well known to the skilled artisan. Generally, they can beadministered to a typical human on a daily basis on an oral dose ofabout 0.1 mg/kg-1000 mg/kg, and more preferably from about 1 mg/kg toabout 200 mg/kg. The parenteral dose will appropriately be 20-100% ofthe oral dose.

Screening Assays

The osteogenic activity of the compounds used in the methods of theinvention can be verified using in vitro screening techniques, such asthe assessment of transcription of a reporter gene coupled to a bonemorphogenetic protein-associated promoter, as described above, or inalternative assays such as the following:

Technique for Neonatal Mouse Calvaria Assay (In vitro)

This assay is similar to that described by Gowen M. & Mundy G. J Immunol(1986) 136:2478-82. Briefly, four days after birth, the front andparietal bones of ICR Swiss white mouse pups are removed bymicrodissection and split along the sagittal suture. The bones areincubated in BGJb medium (Irvine Scientific, Santa Ana, Calif.) plus0.02% (or lower concentration) β-methylcyclodextrin, wherein the mediumalso contains test or control substances, at 37 C in a humidifiedatmosphere of 5% CO₂ and 95% air for 96 hours.

Following this, the bones are removed from the incubation media andfixed in 10% buffered formalin for 24-48 hours, decalcified in 14% EDTAfor 1 week, processed through graded alcohols; and embedded in paraffinwax. Three μm sections of the calvaria are prepared. Representativesections are selected for histomorphometric assessment of bone formationand bone resorption. Bone changes are measured on sections cut 200 μmapart. Osteoblasts and osteoclasts are identified by their distinctivemorphology.

Other auxiliary assays can be used as controls to determine non-BMPpromoter-mediated effects of test compounds. For example, mitogenicactivity can be measured using screening assays featuring aserum-response element (SRE) as a promoter and a luciferase reportergene. More specifically, these screening assays can detect signallingthrough SRE-mediated pathways, such as the protein kinase C pathway. Forinstance, an osteoblast activator SRE-luciferase screen and an insulinmimetic SRE-luciferase screen are useful for this purpose. Similarly,test compound stimulation of cAMP response element (CRE)-mediatedpathways can also be assayed. For instance, cells transfected withreceptors for PTH and calcitonin (two bone-active agents) can be used inCRE-luciferase screens to detect elevated cAMP levels. Thus, the BMPpromoter specificity of a test compound can be examined through use ofthese types of auxiliary assays.

In vivo Assay of Effects of Compounds on Murine Calvarial Bone Growth

Male ICR Swiss white mice, aged 4-6 weeks and weighing 13-26 gm, areemployed, using 4-5 mice per group. The calvarial bone growth assay isperformed as described in PCT application WO 95/24211. Briefly, the testcompound or appropriate control vehicle is injected into thesubcutaneous tissue over the right calvaria of normal mice. Typically,the control vehicle is the vehicle in which the compound wassolubilized, and is PBS containing 5% DMSO or is PBS containing Tween (2μl/10 ml). The animals are sacrificed on day 14 and bone growth measuredby histomorphometry. Bone samples for quantitation are cleaned fromadjacent tissues and fixed in 10% buffered formalin for 24-48 hours,decalcified in 14% EDTA for 1-3 weeks, processed through gradedalcohols; and embedded in paraffin wax. Three to five μm sections of thecalvaria are prepared, and representative sections are selected forhistomorphometric assessment of the effects on bone formation and boneresorption. Sections are measured by using a camera lucida attachment totrace directly the microscopic image onto a digitizing plate. Bonechanges are measured on sections cut 200 μm apart, over 4 adjacent 1×1mm fields on both the injected and noninjected sides of the calvaria.New bone is identified by its characteristic woven structure, andosteoclasts and osteoblasts are identified by their distinctivemorphology. Histomorphometry software (OsteoMeasure, Osteometrix, Inc.,Atlanta) is used to process digitizer input to determine cell counts andmeasure areas or perimeters.

Additional In Vivo Assays

Lead compounds can be further tested in intact animals using an in vivo,dosing assay. Prototypical dosing may be accomplished by subcutaneous,intraperitoneal or oral administration, and may be performed byinjection, sustained release or other delivery techniques. The timeperiod for administration of test compound may vary (for instance, 28days as well as 35 days may be appropriate). An exemplary, in vivosubcutaneous dosing assay may be conducted as follows:

In a typical study, 70 three-month-old female Sprague-Dawley rats areweight-matched and divided into seven groups, with ten animals in eachgroup. This includes a baseline control group of animals sacrificed atthe initiation of the study; a control group administered vehicle only;a PBS-treated control group; and a positive control group administered acompound (non-protein or protein) known to promote bone growth. Threedosage levels of the compound to be tested are administered to theremaining three groups.

Briefly, test compound, positive control compound, PBS, or vehicle aloneis administered subcutaneously once per day for 35 days. All animals areinjected with calcein nine days and two days before sacrifice (twoinjections of calcein administered each designated day). Weekly bodyweights are determined. At the end of the 35-day cycle, the animals areweighed and bled by orbital or cardiac puncture. Serum calcium,phosphate, osteocalcin, and CBCs are determined. Both leg bones (femurand tibia) and lumbar vertebrae are removed, cleaned of adhering softtissue, and stored in 70% ethanol for evaluation, as performed byperipheral quantitative computed tomography (pQCT; Ferretti, J. Bone(1995) 17:353S-64S), dual energy X-ray absorptiometry (DEXA;Laval-Jeantet A. et al. Calcif Tissue Intl (1995) 56:14-18; J. Casez etal. Bone and Mineral (1994) 26:61-68) and/or histomorphometry. Theeffect of test compounds on bone remodeling can thus be evaluated.

Lead compounds also be tested in acute ovariectomized animals(prevention model) using an in vivo dosing assay. Such assays may alsoinclude an estrogen-treated group as a control. An exemplarysubcutaneous dosing assay is performed as follows:

In a typical study, 80 three-month-old female Sprague-Dawley rats areweight-matched and divided into eight groups, with ten animals in eachgroup. This includes a baseline control group of animals sacrificed atthe initiation of the study; three control groups (sham ovariectomized(sham OVX)+vehicle.only; ovariectomized (OVX)+vehicle only; PBS-treatedOVX); and a control OVX group that is administered a compound known topromote bone growth. Three dosage levels of the compound to be testedare administered to the remaining three groups of OVX animals.

Since ovariectomy (OVX) induces hyperphagia, all OVX animals arepair-fed with sham OVX animals throughout the 35 day study. Briefly,test compound, positive control compound, PBS, or vehicle alone isadministered subcutaneously once per day for 35 days. Alternatively,test compound can be formulated in implantable pellets that areimplanted for 35 days, or may be administered orally, such as by gastricgavage. All animals, including sham OVX/vehicle and OVX/vehicle groups,are injected intraperitoneally with calcein nine days and two daysbefore sacrifice (two injections of calcein administered each designatedday, to ensure proper labeling of newly formed bone). Weekly bodyweights are determined. At the end of the 35-day cycle, the animals'blood and tissues are processed as described above.

Lead compounds may also be tested in chronic OVX animals (treatmentmodel). An exemplary protocol for treatment of established bone loss inovariectomized animals that can be used to assess efficacy of anabolicagents may be performed as follows. Briefly, 80 to 100 six month oldfemale, Sprague-Dawley rats are subjected to sham surgery (sham OVX) orovariectomy (OVX) at time 0, and 10 rats are sacrificed to serve asbaseline controls. Body weights are recorded weekly during theexperiment. After approximately 6 weeks of bone depletion (42 days), 10sham OVX and 10 OVX rats are randomly selected for sacrifice asdepletion period controls. Of the remaining animals, 10 sham OVX and 10OVX rats are used as placebo-treated controls. The remaining OVX animalsare treated with 3 to 5 doses of test drug for a period of 5 weeks (35days). As a postitive control, a group of OVX rats can be treated withan agent such as PTH, a known anabolic agent in this model (Kimmel etal. Endocrinology (1993) 132:1577-84). To determine effects on boneformation, the following procedure can be followed. The femurs, tibiaeand lumbar vertebrae 1 to 4 are excised and collected. The proximal leftand right tibiae are used for pQCT measurements, cancellous bone mineraldensity (BMD) (gravimetric determination), and histology, while themidshaft of each tibiae is subjected to cortical BM or histology. Thefemurs are prepared for pQCT scanning of the midshaft prior tobiomechanical testing. With respect to lumbar vertebrae (LV), LV2 areprocessed for BMD (pQCT may also be performed); LV3 are prepared forundecalcified bone histology; and LV4 are processed for mechanicaltesting.

Nature of the Compounds Useful in the Invention

All of the compounds of the invention contain two aromatic systems, Ar¹and Ar², spaced apart by a linker at a distance of 1.5-15 Å, and allgenerally contain at least one nitrogen atom. A summary of thestructural features of the compounds included within the invention isshown in FIG. 1.

As shown, Ar¹ and Ar² may include various preferred embodiments. Theseare selected from the group consisting of a substituted or unsubstitutedaromatic ring system containing a five-membered heterocycle; asubstituted or unsubstituted aromatic ring system containing asix-membered heterocycle; a substituted or unsubstituted naphthalenemoiety; and a substituted or unsubstituted benzene moiety. There are 16possible combinations of these embodiments if Ar¹ and Ar², areconsidered distinguishable. As will be clear, however, the designationof one aromatic system as Ar¹ and the other as Ar² is arbitrary; thusthere are only ten possible combinations. However, for simplicity, Ar¹and Ar² are designated separately with the realization that the choiceis arbitrarily made. All linkers described herein if not palindromic,are considered link Ar¹ to Ar² or vice-versa whether or not thecomplementary orientation is explicitly shown. Thus, if Ar¹ and Ar² aredifferent and a linker is specified as --CONR it is understood that alsoincluded is the linker --NRCO--.

The non-interfering substituents on the aromatic system represented byAr¹ and the non-interfering substituents on the aromatic systemrepresented by Ar² are represented in the formulae herein by R¹ and R²,respectively. Generally, these substituents can be of wide variety.Among substituents that do not interfere with the beneficial effect ofthe compounds of the invention on bone in treated subjects are includedalkyl (1-6C, preferably lower alkyl 1-4C), including straight orbranched-chain forms thereof, alkenyl (1-6C, preferably 1-4C), alkynyl(1-6C, preferably 1-4C), all of which can be straight or branched chainsand may contain further substituents; halogens, including F, Cl, Br andI; siloxy, OR, SR, NR₂, OOCR, COOR, NCOR, NCOOR, and benzoyl, CF₃, OCF₃,SCF₃, N(CF₃)₂, CN, SO, SO₂ R and SO₃ R wherein R is alkyl (1-6C) or isH. Where R¹ or R² substituents are in adjacent positions in the aromaticsystem, they may form a ring. Further, rings may be included insubstituents which contain sufficient carbon and heteroatoms to providethis possibility.

Preferred non-interfering substituents include hydrocarbyl groups of1-6C, including saturated and unsaturated, linear or branchedhydrocarbyl as well as hydrocarbyl groups containing ring systems; halogroups, alkoxy, hydroxy, amino, monoalkyl- and dialkylamino where thealkyl groups are 1-6C, CN, CF₃, and COOR.

Although the number of R¹ and R² may typically be 0-4 or 0-5 dependingon the available positions in the aromatic system, preferred embodimentsinclude those wherein the number of R¹ is 0, 1 or 2 and of R² is 0, 1 or2.

The linker group, L, may be a covalent bond or any group having avalence of at least two and covering a linear distance of from about 1.5to about 15 Angstroms, including those that contain cyclic moieties,that meet this spatial requirement. Useful linkers are divided, bydefinition herein, into three general categories: (1) flexiblenon-conjugating linkers, (2) flexible conjugating linkers, and (3)constrained linkers. The preferred choice of linker will depend on thechoices for Ar¹ and Ar².

As defined herein, flexible non-conjugating linkers are those that linkonly one position of Ar¹ to one position of Ar², and provide only asingle covalent bond or a single chain between Ar¹ and Ar². The chainmay contain branches, but may not contain π-bonds (except in thebranches) or cyclic portions in the chain. The linker atoms in the chainitself rotate freely around single covalent bonds, and thus the linkerhas more than two degrees of freedom. Particularly useful flexiblenon-conjugating linkers, besides a covalent bond, are those of theformulae: --NR--, --CR₂ --, --S--, or --O--, wherein R is H or alkyl(1-6C), more preferably H or lower alkyl (1-4C) and more preferably H.Also preferred are those of the formulae: --NRCO--, --CONR--, --CR₂ S--,--SCR₂ --, --OCR₂ --, --CR₂ O--, --NRNR--, --CR₂ CR₂ --, --NRSO₂ --,--SO₂ NR--, --CR₂ CO--, --COCR₂ --, and --NR--NR--CO--CR₂ -- and itscomplement --CR₂ --CO--NR--NR--, including the isosteres thereof. Alsopreferred are those of the formulae: --NR(CR₂)₂ NR--, --O(CR₂)₂ O--, and--S(CR₂)₂ S--, including the isosteres thereof. The optimum choice oflinker is dependent on the nature of Ar¹ and Ar².

Flexible conjugating linkers are those that link only one position ofAr¹ to one position of Ar², but incorporate at least one double ortriple bond or one or more cyclic systems and thus have only two degreesof freedom. A flexible conjugating linker may form a completelyconjugated π-bond linking system between Ar¹ and Ar², thus providing forco-planarity of Ar¹ and Ar². Examples of useful flexible conjugatinglinkers include: --RC═CR--; --N═N--; --C.tbd.C--; --RC═N--; --N═CR--;--NR--N═CR--; --NR--NR--CO--CR═CR--; and the like, where R is H or alkyl(1-6C); preferably H or lower alkyl (1-4C); and more preferably H.

Constrained linkers are those that have more than one point ofattachment to either or both Ar¹ and Ar² and, thus, generally allow foronly one degree of freedom. Constrained linkers most frequently formfused 5- or 6-membered cyclic moieties with Ar¹ and/or Ar² where eitherAr¹ or Ar² has at least one substituent appropriately positioned to forma second covalent bond with the linker, e.g., where Ar² is a phenylgroup with a reactive, ortho-positioned substituent, or is derivatizedto the linker directly at the ortho position. (Although the aromaticmoieties should properly be refined to as phenylene or naphthylene insuch cases, generally the term "phenyl" or "naphthyl" is used herein toinclude both monovalent and bivalent forms of these moieties.) Examplesof particularly useful constrained linkers include ##STR2## and thelike, where X is O, N, S or CR, and Y is CR₂ or C═O.

All of the compunds of the invention contain constrained linkers.

Synthesis of the Compounds Useful in the Invention

Many of the compounds useful in the invention are commercially availableand can be synthesized by art-known methods. Those compounds useful inthe invention which are new compounds, can similarly be obtained bymethods generally known in the art.

The following examples are intended to illustrate, but not to limit, theinvention.

Preparation A

Compound 59-0008 was synthesized according to the procedure of McDonald,W. S., et al. Chem Comm (1969) 392-393; Irving, H. N. N. H. et al. AnalChim Acta (1970) 49:261-266. Briefly, 10.0 g of dithizone was taken upin 100 ml EtOH and 50 ml AcOH and heated at reflux for 18 h. Aftercooling, this was diluted first with 100 ml water and then with 50 ml 1NNaOH. This was then further neutralized by the addition of 6 N NaOH tobring the pH to 5.0. This deep purple mixture was then concentrated on arotavapor to remove organics. Once the liquid had lost all of its purplecolor, this was filtered to collect the dark precipitate. Purificationby flash chromatography (4.5×25.7 cm; EtAc/Hep. (1:4); R_(f) 0.22)followed by recrystalization from EtOH gave 2.15 g (25% yield) of darkpurple crystals, mp=184-185° C. ¹ H NMR (CDCl₃) 7.90 (d of d, J₁ =7.7,J₂ =2.2, 2H), 7.64 (hump, 1H), 7.49 (m, 3H), 7.02 (m, 1H), 6.91 (m, 2H),6.55 (d, J=8.1, 1H). MS (EI) 254 (47, M+), 105 (26), 77 [100], 51 (27).HRMS (EI, M+) 254.0626 (calcd 254.0626182). Anal. Calcd for C₁₃ H₁₀ N₄S: C, 61.40; H, 3.96; N, 22.03. Found: C, 61.40; H, 4.20; N, 22.06.

EXAMPLE 1 High Throughput Screening

Several thousand compounds were tested in the assay system set forth inU.S. Ser. No. 08/458,434, filed Jun. 2, 1995, and incorporated herein byreference. The standard positive control was a compound of theinvention, 59-0008 (also denoted "OS8"), which is of the formula:##STR3##

In more detail, the 2T3-BMP-2-LUC cells, a stably transformed osteoblastcell line described in Ghosh-Choudhury et al. Endocrinology (1996)137:331-39, referenced above, was employed. The cells were culturedusing α-MEM, 10% FCS with 1% penicillin/streptomycin and 1% glutamine("plating medium"), and were split 1:5 once per week. For the assay, thecells were resuspended in a plating medium containing 4% FCS, plated inmicrotiter plates at a concentration of 5×10³ cells (in 50 μl)/well, andincubated for 24 hours at 37° C. in 5% CO₂. To initiate the assay, 50 μlof the test compound or the control in DMSO was added at 2×concentration to each well, so that the final volume was 100 μl. Thefinal serum concentration was 2% FCS, and the final DMSO concentrationwas 1%. Compound 59-0008 (10 μM) was used as a positive control.

The treated cells were incubated for 24 hours at 37° C. and 5% CO₂. Themedium was then removed, and the cells were rinsed three times with PBS.After removal of excess PBS, 25 μl of 1× cell culture lysing reagent(Promega #E153A) was added to each well and incubated for at least tenminutes. Optionally, the plates/samples could be frozen at this point.To each well was added 50 μl of luciferase substrate (Promega #E152A; 10ml Promega luciferase assay buffer per 7 mg Promega luciferase assaysubstrate). Luminescence was measured on an automated 96-wellluminometer, and was expressed as either picograms of luciferaseactivity per well or as picograms of luciferase activity per microgramof protein.

In this assay, compound 59-0008 (3-phenylazo-1H-4,1,2-benzothiadiazine)exhibited a pattern of reactivity, as shown in FIG. 2. The activity forcompound 59-0008 was maximal at a concentration of approximately 3-10 μMand, more particularly, at about 3 μM, and thus provided a response ofapproximately 175 light emission units. Accordingly, other testedcompounds were evaluated at various concentrations, and these resultswere compared to the results obtained for 59-0008 at 10 μM (which valuewas normalized to 100). For instance, any tested compound in FIG. 3 andFIG. 4 that showed greater activity than 10 μM of 59-0008 would resultin a value over 100.

As shown in FIG. 3 (46 sheets) and FIG. 4 (28 sheets), several compoundswere found to be particularly effective.

EXAMPLE 2 In vivo Calvarial Bone Growth Data

Compound 59-0008 was assayed in vivo according to the proceduredescribed previously (see "In vivo Assay of Effects of Compounds onMurine Calvarial Bone Growth", supra). As compared to a vehicle control,compound 59-0008 induced a 4-fold increase in width of new calvarialbone.

EXAMPLE 3 Chondrogenic Activity

Compounds 59-008, 59-0102 and 50-0197 were assayed for effects on thedifferentiation of cartilage cells, as compared to the action ofrecombinant human BMP-2. Briefly, a mouse clonal chondrogenic cell line,TMC-23, was isolated and cloned from costal cartilage of transgenic micecontaining the BMP-2 gene control region driving SV-40 large T-antigen,generated as described in Ghosh-Choudhury et al Endocrinology137:331-39, 1996. These cells were cultured in DMEM/10% FCS, and wereshown to express T-antigen, and also to produce aggrecan (toluidine bluestaining at pH 1.0) and Type-II collagen (immunostaining) by 7 daysafter confluence.

For measurement of alkaline phosphatase (ALP) activity, the technique ofLF Bonewald et al. J Biol Chem (1992) 267:8943-49, was employed.Briefly, TMC-23 cells were plated in 96 well microtiter plates in DMEMcontaining 10% FCS at 4×10³ cells/well. Two days after plating, thecells were confluent and the medium was replaced with fresh mediumcontaining 10% FCS and different concentrations of compounds orrecombinant BMP-2. After an additional 2 or 5 days incubation, theplates were washed twice with PBS, and then lysing solution (0.05%Triton X-100) was added (100 μl/well). The cells were lysed by threefreeze-thaw cycles of -70° C. (30 min), followed by 37° C. (30 min withshaking). Twenty microliters of cell lysates were assayed with 80 μl of5 mM p-nitrophenol phosphate in 1.5 M 2-amino-2-methyl-propanol buffer,pH 10.3 (Sigma ALP kit, Sigma Chemical Co., St. Louis, Mo.) for 10 minat 37° C. The reaction was stopped by the addition of 100 μl of 0.5 MNaOH. The spectrophotometric absorbance at 405 nm was compared to thatof p-nitrophenol standards to estimate ALP activity in the samples. Theprotein content of the cell lysates was determined by the Bio-Radprotein assay kit (Bio-Rad, Hercules, Calif.). Specific activity wascalculated using these two parameters.

At day 2, compounds 59-0008 (10⁻⁹ M), 59-0102 (10⁻⁷ M) and 59-0197 (10⁻⁹M) increased ALP levels approximately 3-, 2- and 2.5-fold, respectively,as compared to the vehicle control. Recombinant BMP2 at 100, 50 or 10ng/ml induced ALP levels approximately 10-, 4- or 1.5-fold,respectively, as compared to the vehicle control.

EXAMPLE 4 Synthesis of Exemplary Compounds

Constrained analogs having the linker depicted below: ##STR4## may besynthesized by reference to the methods described in Gorbulenko, N. V.et al. Dokl Akad Nauk Ukr SSR (1991) 5:117-23, substituting the6-membered heterocycle for benzothiazole.

Related, constrained analogs having the linker depicted below: ##STR5##may be synthesized by reference to the methods described in thefollowing publications: Chaurasia, M. R. & Sharma, A. J. Acta CiencIndica Chem (1992) 18:419-22; Kandeel, Maymona M., in Phosphorus,Sulfur, Silicon, Relat Elem (1990) 48:149-55; Salem, M. A. & Soliman, E.A. Egypt J Chem (1985) 27:779-87;Garin, J. et al. Synthesis (1984)6:520-22, and Ayyangar N. R. et al. Dyes and Pigments (1990) 13:301-10.

We claim:
 1. A method to treat a condition in a vertebrate animalcharacterized by a deficiency in, or need for, bone growth replacementand/or an undesirable level of bone resorption, which method comprisesadministering to a vertebrate subject in need of such treatment aneffective amount of a compound of the formula: ##STR6## wherein R¹ is anon-interfering substituent; m is an integer of 0-4;each dotted linerepresents an optional π-bond; each Z is independently N, NR, O, S, CRor CR₂, where each R is independently H or alkyl (1-6C); L is aconstrained linker; and Ar² is a bivalent phenyl, bivalent napthyl, abivalent aromatic system containing a 6-membered ring or a bivalentaromatic system containing a 5-membered ring, all of which may besubstituted or unsubstituted.
 2. The method of claim 1 wherein Ar¹ is##STR7## .
 3. The method of claim 1 wherein Ar² is ##STR8## where R² isa non-interfering substituent and m is an integer from 0 to
 4. 4. Themethod of claim 1 wherein Ar² is unsubstituted phenylene.
 5. The methodof claim 1 wherein at least one Z is N or NR.
 6. The method of claim 5wherein at least two Z are N or NR.
 7. The method of claim 6 wherein allof Z are N or NR.
 8. The method of claim 5 wherein at least oneremaining Z is CR or CR₂.
 9. The method of claim 8 wherein bothremaining Z are CR or CR₂.
 10. The method of claim 5 wherein at least one remaining Z is O or S.
 11. The method of claim 10 wherein bothremaining Z are O or S.
 12. The method of claim 6 wherein the remainingZ is CR or CR₂.
 13. The method of claim 6 wherein the remaining Z is Oor S.